Cyclic ADP-ribose signal transduction : Molecular mechanism and pathophsiological significance

环状ADP-核糖信号转导：分子机制和病理生理学意义

• 批准号: 08102003
• 负责人: OKAMOTO Hiroshi
• 金额: $ 184.32万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Specially Promoted Research
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08102003/
• 关键词: CD38; cyclic ADP-ribose; ADP-ribosyl cyclase; cyclic ADP-ribose hydrolase; second messenger; Ca^<2+> release channel; insulin secretion; molecular cloning; Ca^<2+>放出チャンネル

Calcium mobilization plays a central role in a variety of cellular responses such as insulin secretion by glucose in pancreatic β-cells. We have recently found that cyclic ADP-ribose (cADPR), a metabolite of NAD^+, is a novel second messenger for Ca^<2+> mobilization for insulin secretion. In the project, we have elucidated the molecular mechanism of cADPR signal transduction in mammalian cells.1. We identified the binding sites for NAD^+ (substrate), cADPR (product) and ATP (regulator) for CD38 (mammalian ADP-ribosyl cyclase/cADPR hydrolase) by site-directed mutagenesis.2. We isolated the human CD38 gene and determined its primary structure. The gene consists of 8 exons that extend -100 kbp on band p15 of chromosome 4 as a single copy gene. We found an Arg140→Trp mutation in Japanese NIDDM patients and the mutated protein exhibited a decrease in the enzymic activity of CD38 to form cADPR.3. We found autoantibodies against CD38 that impair glucose-induced insulin secretion in Japanese … More and Caucasian subjects with diabetes.4. We demonstrated that cADPR binds to FK506-binding protein 12.6 (FKBP12.6) on rat islet ryanodine receptor (RyR) and that the binding of cADPR to FKBP12.6 frees the RyR from FKBP12.6, releasing Ca^<2+>. In addition, in heart failure, the stoichiometry of FK506-binding protein per the ryanodine receptor was decreased. We isolated the human FKBP12.6 gene and determined the primary structure. The gene spans about 16 kbp on chromosome 2p21-23.5. We found that the Ca^<2+> release from microsomes was greatly enhanced by the activation of CaM kinase II and A-kinase.6. We produced knockout mice carrying a null mutation in the CD38 gene by homologous recombination and found that CD38 disruption impairs glucose-induced increases in cADPR, intracellular Ca^<2+> concentration, and insulin secretion.7. Using CD38 knockout mice, we found that muscarinic Ca^<2+> signaling in pancreatic acinar cells involves a CD38-dependent pathway responsible for two cADPR-dependent Ca^<2+> release mechanisms in which the one sensitive to ryanodine plays a crucial role for the generation of repetitive Ca^<2+> spikes.8. We determined the primary structure of streptococcal ADP-ribosyl cyclase/cADPR hydrolase. Using sitedirected mutagenesis, we found that Lys-162 and/or Lys-163 of the streptococcal enzyme, which correspond to Lys-129 of human CD38, participate in the cADPR binding and that Glu-307 of the streptococcal enzyme and Glu-226 of human CD38 are essential for the NAD^+ binding.9. We found type 2 and type 3 ryanodine receptor Ca^<2+> channel (RyR) is expressed in normal pancreatic β-cells and the expression is markedly reduced in diabetic β-cells. We have made mutant mice lacking the both types of RyR gene in insulin producing β-cells. Less

在胰腺β细胞中，钙动员在多种细胞反应中起着核心作用，如葡萄糖分泌胰岛素。我们最近发现，NAD^+的代谢产物环状ADP-核糖(CADPR)是一种新的动员Ca~(2+)和GT；分泌胰岛素的第二信使。在本项目中，我们阐明了cADPR在哺乳动物细胞中信号转导的分子机制。我们用定点突变的方法确定了CD38(哺乳动物ADP-核糖环化酶/cADPR水解酶)与NAD^+(底物)、cADPR(产物)和ATP(调节子)的结合部位。我们分离了人CD38基因，并测定了其一级结构。该基因由8个外显子组成，这些外显子在4号染色体的p15带上延伸-100kbp，为单拷贝基因。我们在日本NIDDM患者中发现了Arg140CD38Trp突变，突变的蛋白显示→酶活性降低以形成cADPR3。我们在日本…中发现了抗CD38的自身抗体能抑制葡萄糖诱导的胰岛素分泌糖尿病患者多为高加索人。我们证明了cADPR与大鼠胰岛Ryanodine受体(RyR)上的FK506结合蛋白12.6(FKBP12.6)结合，cADPR与FKBP12.6结合使RyR从FKBP12.6中释放出来，释放出Ca~(2+)和Gt；此外，在心力衰竭时，兰尼定受体上FK506结合蛋白的化学计量比降低。我们分离了人FKBP12.6基因，并测定了其一级结构。该基因位于染色体2p21-23.5上，大小约为16kbp。我们发现，CaM激酶II和A-Kinase的激活显著促进了微生物体的Ca~(2+)释放。我们通过同源重组产生了CD38基因零突变的基因敲除小鼠，发现CD38基因的中断损害了葡萄糖诱导的cADPR、细胞内钙离子浓度和胰岛素分泌的增加。利用CD38基因敲除的小鼠，我们发现胰腺腺泡细胞中的M胆碱钙信号涉及CD38依赖的通路，该通路负责两种依赖cADPR的钙释放机制，其中对兰诺定敏感的钙离子在重复的钙尖峰的产生中起关键作用。我们测定了链球菌ADP-核糖环化酶/cADPR水解酶的一级结构。利用定点突变，我们发现链球菌酶的Lys-162和/或Lys-163，对应于人CD38的Lys-129，参与了cADPR的结合，链球菌酶的Glu-307和人CD38的Glu-226是NAD^+结合所必需的。我们发现2型和3型Ryanodine受体钙通道(RyR)在正常胰腺β-细胞中有表达，在糖尿病β-细胞中的表达明显降低。我们已经在产生胰岛素的β细胞中获得了缺乏这两种类型RyR基因的突变小鼠。较少

项目成果

Kazumori,H.: "Neutrophil chemoattractant-2βregulates the expression of the Reg gene in injured gastric mucosa in rats."Gastroenterology. 119. 1610-1622 (2000)

Kazumori, H.：“中性粒细胞趋化剂-2β 调节大鼠受损胃粘膜中 Reg 基因的表达。胃肠病学”119. 1610-1622 (2000)。

Akiyama,T.: "Activation of Reg gene, a gene for insulin-producing β- cell regeneration: Poly (ADP-ribose) polymerase binds Reg promoter and regulates the transcription by autopoly (ADP-ribosyl)ation."Proc. Natl. Acad. Sci. USA. 98. 48-53 (2001)

Akiyama，T.：“Reg 基因的激活，一种用于产生胰岛素的 β 细胞再生的基因：聚（ADP-核糖）聚合酶结合 Reg 启动子并通过自聚（ADP-核糖基）化来调节转录。”Proc。美国科学。98。48-53（2001）

小林誠一: "インスリン産生細胞増殖因子Reg受容体の構造・発現・機能"生化学. 73・2. 132-132 (2001)

小林精一：“胰岛素生成细胞生长因子Reg受体的结构、表达和功能”生物化学73・2（2001）。

Seiichi Kobayashi: "Identification of a receptor for Reg (Regenerating Gene) protein, a pancreatic beta-cell regeneration factor."Journal of Biological Chemistry. 275. 10723-10726 (2000)

Seiichi Kobayashi：“Reg（再生基因）蛋白受体的鉴定，这是一种胰腺β细胞再生因子。”生物化学杂志。

Masafumi Yano: "Altered stoichiomrtory of FKBP12.6 versus ryanodine receptor as a cause of abnormal Ca^<2+> leak through ryanodine receptor in heart failure."Circulation. 102. 2131-2136 (2000)

Masafumi Yano：“FKBP12.6 与兰尼碱受体相比化学计量的改变是心力衰竭中通过兰尼碱受体异常 Ca^2 渗漏的原因。”循环。